CN103159854A - Connection method of antibody and microsphere - Google Patents
Connection method of antibody and microsphere Download PDFInfo
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- CN103159854A CN103159854A CN2013100729394A CN201310072939A CN103159854A CN 103159854 A CN103159854 A CN 103159854A CN 2013100729394 A CN2013100729394 A CN 2013100729394A CN 201310072939 A CN201310072939 A CN 201310072939A CN 103159854 A CN103159854 A CN 103159854A
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- GBMSZXWHMSSBGP-UHFFFAOYSA-N CC(C)C(C)CN Chemical compound CC(C)C(C)CN GBMSZXWHMSSBGP-UHFFFAOYSA-N 0.000 description 1
- 0 CC(c1cnc(*)cc1)=O Chemical compound CC(c1cnc(*)cc1)=O 0.000 description 1
- JESQEXHNTFKPIP-UHFFFAOYSA-N [O-][NH+](c1ccc(C=O)cc1)O Chemical compound [O-][NH+](c1ccc(C=O)cc1)O JESQEXHNTFKPIP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a connection method of antibody and microsphere, which comprises crosslinking of the antibody and nucleotide, combination of nucleotide and microsphere, and hybridization of a nucleotide complementary chain. The crosslinking of the antibody and nucleotide comprises modification and desalination of the antibody, modification and desalination of nucleotide, crosslinking and purifying of the modified antibody and nucleotide, and the like. The connection method takes a double functional group of nucleotide and the antibody as modification, and the nucleotide and the antibody are combined through an aldehyde group and an amino group to form hydrazone bond combination. According to the invention, the combination of the antibody and microsphere is completed by microsphere coated with avidin and biotin which is modified on a nucleotide chain through specifically adsorption; the hybridization of the nucleotide complementary chain is completed under certain temperature condition through a specific hybridization liquid; the incubation and identification for whether the reaction on the microsphere successes or not can be carried out under the specific temperature condition through Cy3 labeled secondary antibody and the like. The connection method takes oligonucleotide as a support arm for separating the antibody and the magnetic bead for a certain distance, and is in favor of avoiding the destroy of a structure of the antibody, and thereby the antibody activity can be protected.
Description
Technical field
The present invention relates to a kind of antibody and microballon method of attachment.
Background technology
From 1979, polystyrene microbeads successfully magnetized and with after antibody is connected, namely become the splendid separation method of a kind of effect, having caused the revolution on the bioseparation technology.The method has the following advantages: (1) velocity of separation is fast, efficient is high, favorable repeatability; (2) simple to operate, do not need expensive plant and instrument; (3) specific surface area is large, can improve detection sensitivity; (4) do not affect biological character and the function of separated cell or other biomaterial.
Along with the development of science and technology and the popularization of nanotechnology, nano material has been brought into play vital role in the life science field.The magnetic Nano microballon has been successfully applied in the systems such as nucleic acid extraction, wastewater treatment, targeted drug, food safety detection, and has obtained certain progress as a kind of novel nanotechnology.The progress of coating technique can be expanded the range of application of magnetic micro-beads greatly.Therefore, interconnection technique and the method for research bead surface will be used to more wide separation, detection field it, and will greatly be improved detection sensitivity.
Food safety is healthy concerning broad masses of the people's, is subject to showing great attention to of government and common people.The food quality problem is very outstanding in recent years, illegally uses forbidden drug, abuse of antibiotics, and excess Use overrun veterinary drug etc. have all caused very large harm to the masses are healthy.For above problem, to strengthen supervision on the one hand; To develop specific sharp separation, detection method on the other hand.To be a kind of comparatively easy and efficient context of detection to the specific antibody of magnetic micro-beads pan coating.Method for coating is various, and comparatively commonly used is that antibody directly is fixed to bead surface by the group effect.Yet the sensitivity of this kind method is limited, and the non-specific adsorption of antibody on microballon will greatly reduce it and detect effect.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of antibody and microballon method of attachment that can improve detection sensitivity.
In order to solve the problems of the technologies described above, the invention provides a kind of antibody and microballon method of attachment, comprise the following steps:
1), antibody and Nucleotide are crosslinked:
The modification of A, antibody and desalination:
25～100 μ g immunoglobulin (Ig) (Immunoglobulin, be called for short IgG) be diluted to concentration be after 2～8mg/mL with SANH(Succinimidyl 6-hydrazinonicotinate acetone hydrazone) evenly mix after, hatch in the shaking table of 20～30 ℃ and modified 2～6 hours, the mol ratio of SANH and immunoglobulin (Ig) is 10～70:1;
With the antibody modification product of gained through washing, centrifuging, to remove unnecessary SANH; The antibody modification product after desalination; Survey the protein concentration of IgG, stand-by (purpose is: the rate of recovery of antibody after calculating modification, desalination step, avoid because ultra-filtration membrane or operational issue cause sample too to lose.）
The modification of B, Nucleotide and desalination:
Phosphoric acid buffer with pH7.0～7.6 is diluted to 6～12mg/mL with Nucleotide (oligonucleotide), add SFB(Succinimidyl 4-formylbenzoate) evenly mix, hatch in the shaking table of 20～30 ℃ and modified 2～6 hours, the mol ratio of SFB and Nucleotide (oligonucleotide) is 10～70:1;
Remarks explanations: above-mentioned nucleotides sequence is classified GGGAA ATCTC TGCAG GCAAA TGTGA(5 '～3 ' as), the 3 ' terminal modified amino;
With the nucleotide modification product of gained through washing, centrifugal, filter, to remove unnecessary SFB; The nucleotide modification product after desalination; Survey nucleotide concentration, stand-by (purpose is the rate of recovery of oligonucleotide after calculating modification, desalination step, avoids because ultra-filtration membrane or operational issue cause sample too to lose.）
Crosslinked and the purifying of antibody and Nucleotide after C, modification
Nucleotide modification product after antibody modification product after desalination (for modifying the antibody modification product after successful desalination) and desalination (be the successful desalination of modification after nucleotide modification product) is evenly mixed according to the mol ratio of 1:8～12, fully reacted 8～24 hours in 2～8 ℃
With the crosslinked after product of gained through washing, centrifugal, filter, remove free oligonucleotide and carry out desalination; The antibody-Nucleotide crosslinked after purifying;
Measure light absorption value under 350nm, stand-by (modification group of antibody and oligonucleotide generates the hydrazone key of stable existence in crosslinked process, this hydrazone key has special absorption peak under 350nm; Therefore whether measuring 350nm, to have obvious absorption peaks to generate be the crosslinked successful strong proof of checking; By measuring special absorption peak under 350nm, can further calculate the concentration of cross-linking products);
2), antibody-Nucleotide crosslinked and microballon effect:
Microballon is first through the washings washing, and is then standing;
With 1～9 * 10
-11(sequence is TCACA TTTGC CTGCA GAGAT TTCCC5 '～3 ' to the Nucleotide that mol biotin modifies, 3 ' biotin modification) add the coated magnetic bead of Neutravidin of 5～15 μ l, hatched 10～30 minutes in the shaking table of 20～30 ℃, then wash with washings; Add subsequently above-mentioned 5～10 * 10
-13Antibody after the mol purifying-Nucleotide crosslinked is hatched 1～4 hour in the shaking table of 20～30 ℃, then is washed with washings under 6 * SSPE damping fluid (pH6.0) condition; Get the microballon system;
Described washings is 0.1～0.5% to get for BSA is diluted to mass concentration with PBS solution (pH7.4).
The remarks explanation:
Nucleotide modification product after the desalination of the antibody modification product after the desalination of steps A gained and step B gained identifies by paranitrobenzaldehyde and 2-hydrazine pyridine dihydrochloride whether base group modification is successful respectively; Method after for the desalination that respectively takes a morsel the antibody modification product or the nucleotide modification product after desalination, add respectively excessive paranitrobenzaldehyde, 2-hydrazine pyridine dihydrochloride, after lucifuge is hatched, measure the light absorption value of product group under specific wavelength.Special absorption peak is arranged under 390nm when the Identification of the antibodies product, judge and modify successfully; Identify that when oligonucleotide product has special absorption peak under 350nm, be judged to be and modify successfully.
Improvement as antibody of the present invention and microballon method of attachment:
The microballon system of gained is carried out following steps:
Add the microballon system at the fluorescence two of Cy3 mark in anti-or transgene protein, shaking table was hatched 1～4 hour under 20～30 ℃ of conditions; Then use PBS(pH7.4) wash away the anti-or transgene protein of unconjugated fluorescence two, use at last the fluorescence microscope result.
Remarks explanation: observe fluorescent signal under fluorescent microscope, prove that antibody successfully has been connected to the surface of magnetic bead by " contact " of oligonucleotide.
Further improvement as antibody of the present invention and microballon method of attachment:
The anti-volume ratio with PBS damping fluid (pH7.4) of fluorescence two of Cy3 mark is: 1:10～30.
Further improvement as antibody of the present invention and microballon method of attachment:
In step 1) A: the rotating speed of shaking table is 50～150 rev/mins; EDTA solution with the 0.1mM of pH8 washs;
In step 1) B: the rotating speed of shaking table is 50～150 rev/mins; EDTA solution washing with the 0.1mM of pH8;
In step 1) C: the EDTA solution washing of the crosslinked after product of gained being used the 0.1mM of pH8.
In the A of step 1):
Hatched rear collection modified outcome, product is moved into the YM-100(molecular weight cut-off of Mi Libo greater than the biomolecules of 10000KDa) chimney filter the inside, with EDTA solution washing, the centrifuging of the 0.1mM of pH8 2～4 times, remove unnecessary SANH modifier, then the back-off chimney filter is centrifugal, the modified outcome after the collection desalination.Survey the protein concentration of IgG, stand-by.
In the B of step 1):
Hatched rear collection modified outcome, product is moved into the YM-3(molecular weight cut-off of Mi Libo greater than the biomolecules of 3000KDa) chimney filter the inside, with EDTA solution washing, the centrifuging of the 0.1mM of pH8 2～4 times, remove unnecessary SFB modifier, then the back-off chimney filter is centrifugal, the nucleotide modification product after the collection desalination.Survey nucleotide concentration, stand-by.
In the C of step 1):
Because crosslinked after product needs purifying again, the crosslinked after product of crosslinked complete rear collection, product is moved into the YM-100(molecular weight cut-off of Mi Libo greater than the biomolecules of 10000KDa) chimney filter the inside, with 0.1mM EDTA solution washing, the centrifuging of pH8 2～4 times, remove uncrosslinked unnecessary nucleotide monomer, then the back-off chimney filter is centrifugal, cross-linking products after the collection purifying.Measure light absorption value under 350nm, stand-by.
In the present invention, any one antibody all can be fixed on microballon; With antibody linked oligonucleotide length generally 20-23 base, the 3 ' terminal modified amino, thus can be by method of the present invention and antibody linked.The magnetic bead of selecting has been coated with avidin in advance; The oligonucleotide that is connected with magnetic bead surfaces must with antibody linked oligonucleotide corresponding complementary, and terminal modified vitamin H is arranged 3 ', thereby can be connected with magnetic bead surfaces as method of the present invention.
Particularly, the present invention has developed a kind of method that novel antibody is connected with microballon, and successful antagonist and Nucleotide carry out crosslinked, and keep its excellent activity.Method of attachment of the present invention be with the complementary nucleotide chain of vitamin H (biotion) be coated with after avidin (Neutravidin) microballon is connected, mode by two nucleotide chain hybridization, antibody-Nucleotide crosslinked is connected to bead surface completes being connected of antibody and microballon, at last with whether successful connection of two anti-evaluations of Cy3 mark.Concrete principle as shown in Figure 1.
Antibody of the present invention and microballon cross-linking method: comprise that antibody and Nucleotide are crosslinked, Nucleotide is combined with microballon, the flow processs such as Nucleotide complementary strand hybridization.Crosslinked modification and desalination, the modification of Nucleotide and the steps such as crosslinked and purifying of desalination, the rear antibody of modification and Nucleotide that comprises antibody of antibody and Nucleotide.The present invention is take bifunctional group as Nucleotide and antibody is modified, and by aldehyde radical and amino, it is closed in conjunction with forming the hydrazone bond.In the present invention, the combination of Nucleotide and microballon is to be completed by the vitamin H specific adsorption of modifying on avidin coated on microballon and nucleotide chain; The hybridization of Nucleotide complementary strand is completed under the certain temperature condition by specific hybridization solution; Resist to hatch to identify whether successfully to react on microballon under specific temperature conditions by two of Cy3 mark.
Antibody of the present invention and microballon method of attachment have following advantage:
1, oligonucleotide conduct " sway brace ", spaced apart with antibody and magnetic bead surfaces, is conducive to avoid antibody to suffer structure deteriorate, the protection antibody activity.
2 active groups that solved when antibody is fixed in solid phase carrier spread out to problem.
3, improve the handiness of antibody and other substance reactions.
Description of drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is antibody and microballon cross-linking method schematic diagram.
Fig. 2 is antibody and the anti-evaluation image of the microballon crosslinked rear fluorescence two of success.
Embodiment
Embodiment 1, rabbit immunoglobulin (IgG) are connected with magnetic bead:
1), rabbit immunoglobulin (IgG) is crosslinked with Nucleotide
Modification and the desalination of A, rabbit immunoglobulin (IgG)
With the rabbit immunoglobulin of 50 μ g (Rabbit Immunoglobulin is called for short Rabbit IgG) with modify damping fluid (100mM phosphoric acid salt, 150mM sodium-chlor, pH7.2-7.4) being diluted to concentration is 6mg/mL; Then (concentration is 6.89 * 10 to use SANH solution
-2Mol/L) modify, the mol ratio of SANH and Rabbit IgG is 60:1.SANH solution with after Rabbit IgG solution evenly mixes, is positioned in the shaking table of 25 ℃, hatches with the rotating speed of 100 rev/mins and modified 4 hours.After having hatched, product is moved into the YM-100 chimney filter the inside of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (5000 rev/mins) filter 23, to remove unnecessary SANH modifier, back-off chimney filter centrifugal (5000 rev/mins) is then collected the antibody modification product after desalination.The protein concentration of surveying Rabbit IgG is 2.44mg/mL, and is stand-by.
Antibody modification product after the desalination of above step gained is taken a morsel, by identifying base group modification whether successfully (reacting as follows) with paranitrobenzaldehyde (4-nitrobenzaldehyde) reaction;
Operation steps:
(1) reaction group and control group are set, get 1 μ L and modify rear antibody-solutions in the reaction group, add 5 μ L4-nitrobenzaldehyde(0.5mM) mix; Get the blank damping fluid of modifying of 1 μ L in control group, add 5 μ L4-nitrobenzaldehyde(0.5mM) mix, all be placed in 37 ℃, lucifuge, reaction 30min with two groups.
(2) carry out UV with Nanodrop and measure, with control group as " blank " after, the UV absorption spectrum of assaying reaction group obtains 390nm place absorption peak.Occur if any special absorption peak, can be judged to be and modify successfully.If there is no special absorption peak, can't determine whether and modify successfully, need to again optimize modifying method.But to certain specific antibody sample (Rabbit IgG etc.) but during ripe and repetitive operation of modifying method, can omit the step of evaluation for saving sample.
Remarks explanation: SANH, Succinimidyl4-hydrazinonicotinate acetone hydrazone, that is, and succinimido 4-diazanyl nicotinate acetone hydrazone.Available from the S-HyNic(SANH of U.S. SoluLink company) Kit article No. (S-9002-2).Product was Powdered originally, used DMF(N, N-Dimethylformamide, N-DMF) dissolution with solvents is solution.
The modification of B, Nucleotide and desalination
Nucleotides sequence is classified GGGAA ATCTC TGCAG GCAAA TGTGA(5 '～3 ' as), the 3 ' terminal modified amino.PBS with pH7.4 is diluted to 10mg/mL with Nucleotide, and (concentration is 8.094 * 10 then to use SFB solution
-2MM) modify.Mol ratio between SFB and Nucleotide is 60:1.SFB solution with after Nucleotide solution evenly mixes, is positioned in the shaking table of 25 ℃, hatches with the rotating speed of 100 rev/mins and modified 4 hours.Hatched product has been moved in the YM-3 chimney filter of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (13000 rev/mins) filter 23, to remove unnecessary SFB modifier, then back-off chimney filter centrifugal (13000 rev/mins), nucleotide modification product after the collection desalination, the concentration that records Nucleotide is 3-4ug/Ul3.42ug/uL; Stand-by.
With the nucleotide modification product after the desalination of above-mentioned steps gained by identifying whether success (reacting as follows) of base group modification with 2-hydrazine pyridine dihydrochloride (2-hydrazinopyridine);
Operation steps:
(1) reaction group and control group are set, get 1 μ L and modify rear oligonucleotide solution in the reaction group, add 5 μ L2-hydrazinopyridine(0.5mM) mix; Get the blank damping fluid of modifying of 1 μ L in control group, add 5 μ L2-hydrazinopyridine(0.5mM) mix, all be placed in 37 ℃, lucifuge, reaction 30min with two groups.
(2) carry out UV with Nanodrop and measure, with control group as " blank " after, the UV absorption spectrum of assaying reaction group obtains 350nm place absorption peak.Occur if any special absorption peak, can be judged to be and modify successfully.
Remarks explanation: SFB:Succinimidyl 4-formylbenzoate, i.e. succinimido 4-formyl radical benzoic ether.Available from the S-HyNic(SANH of U.S. SoluLink company) Kit article No. (S-9002-2).Product was Powdered originally, used DMF(N, N-Dimethylformamide, N-DMF) dissolution with solvents is solution.
Crosslinked and the purifying of rabbit immunoglobulin (IgG) and Nucleotide after C, modification
To fully react 12 hours under identifying also after desalination that modifying successful antibody (i.e. antibody modification product after successful desalination is modified in judgement) evenly mixes with the 1:10 mol ratio with oligonucleotide (the nucleotide modification product after successful desalination is modified in judgement), placing 4 ℃.The group of its formation has obvious absorption peak under the 350nm wavelength, record OD
350=0.083, prove crosslinked success.
Crosslinked after product needs purifying again, it is moved into the YM-100 chimney filter the inside of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (5000 rev/mins) filter 23, to remove uncrosslinked unnecessary nucleotide monomer, back-off chimney filter centrifugal (5000 rev/mins) is collected.Product is measured light absorption value under 350nm, obtain the cross-linking products concentration after purifying, in the situation that the less and system volume of loss is consistent, front roughly the same of the crosslinked concentration after purifying and purifying, or (being 1.28mg/mL in the present embodiment) slightly on the low side.
2), antibody-Nucleotide crosslinked and microballon effect:
A, microballon are connected with complementary nucleotide
It is 0.1～0.5% that BSA is diluted to mass concentration with PBS solution (pH7.4); As washings, the coated microballon of Neutravidin is washed (totally 3 times), standing 20 minutes.With 2 * 10
-11(sequence is TCACA TTTGC CTGCA GAGAT TTCCC5 '～3 ' to the Nucleotide that mol biotin modifies, 3 ' biotin modification) add the coated magnetic bead of 5 μ l Neutravidin, 25 ℃ of shaking tables were hatched 15 minutes, with above-mentioned washings washing 3 times, obtained the coated magnetic bead of Nucleotide.
Remarks: microballon can obtain by commercial form, for example can be available from the UniQ magnetic beads of BioQ Technologies company, and coated Neutravidin, diameter 300nm.
B, antibody-Nucleotide crosslinked are connected with microballon
Get approximately 0.1 μ l(namely 8.5 * 10 of antibody-Nucleotide crosslinked
-13Cross-linking products after the purifying of step 1 gained of mol) add the coated magnetic bead of above-mentioned Nucleotide, shaking table is hatched 2 hours (25 ℃) under 6 * SSPE damping fluid (pH6.0) condition, with above-mentioned washings washing 3 times.
Experiment 1, magnetic bead surfaces connect antibody to two anti-the catching of Cy3 fluorescent mark
1 μ l Cy3 fluorescent mark two is anti-with PBS(pH7.4) the 1:20 dilution, then add microballon system (being the product of above-mentioned steps " antibody-Nucleotide crosslinked and microballon effect " gained), shaking table was hatched 2 hours under 25 ℃ of conditions.Then use PBS(pH7.4) washing, purpose is anti-in order to wash away unconjugated fluorescence two.Use at last the fluorescence microscope result, the antibody of confirmation bead surface is successfully caught two and is resisted.As shown in Figure 2:
Observe with fluorescent microscope (Nikon, Inc) under the 510-560 excitation wavelength, be evenly distributed with the bright spot of star spot shape in the visual field.This result proof antibody successfully is connected to magnetic bead surfaces, and it is anti-to catch Cy3 fluorescent mark two.
Embodiment 2 transgenosis Cry1Ac protein monoclonal antibodies are connected with magnetic bead surfaces
1), transgenosis Cry1Ac protein monoclonal antibody and Nucleotide are crosslinked
The modification of A, antibody and desalination
Get the transgenosis Cry1Ac protein monoclonal antibody (preparation of Hangzhou Epitomics Inc.) of 50 μ g, concentration is adjusted into 4mg/mL.With the SANH powder (Succinimidyl4-hydrazinonicotinate acetone hydrazone, that is, succinimido 4-diazanyl nicotinate acetone hydrazone.Available from the S-HyNic(SANH of U.S. SoluLink company) Kit article No. (S-9002-2)) use DMF(N, N-Dimethylformamide, N-DMF) dissolution with solvents is that (concentration is 6.89 * 10 to solution
-2Mol/L) modify.The SANH solution of getting 60 times of molar weights of antibody mixes with antibody-solutions, is positioned in the shaking table of 25 ℃, hatches with the rotating speed of 100 rev/mins and modifies 4 hours.After having hatched, product is moved into the YM-100 chimney filter the inside of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (5000 rev/mins) filter 23, to remove unnecessary SANH modifier, then back-off chimney filter centrifugal (5000 rev/mins), antibody modification product after the collection desalination is about 20 μ L.The survey antibody concentration is 2.68mg/mL, and is stand-by.
The modification of B, Nucleotide and desalination
Nucleotides sequence is classified GGGAA ATCTC TGCAG GCAAA TGTGA(5 '～3 ' as), the 3 ' terminal modified amino.PBS with pH7.4 is diluted to 10mg/mL with Nucleotide.With SFB powder (Succinimidyl 4-formylbenzoate, i.e. succinimido 4-formyl radical benzoic ether.Available from the S-HyNic(SANH of U.S. SoluLink company) Kit article No. (S-9002-2)) use DMF(N, N-Dimethylformamide, N-DMF) dissolution with solvents is solution, making concentration is 8.094 * 10
-2MM.Mol ratio between SFB and Nucleotide is 60:1.SFB solution with after Nucleotide solution evenly mixes, is positioned in the shaking table of 25 ℃, hatches with the rotating speed of 100 rev/mins and modified 4 hours.Hatched product has been moved in the YM-3 chimney filter of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (13000 rev/mins) filter 23, to remove unnecessary SFB modifier, then back-off chimney filter centrifugal (13000 rev/mins), nucleotide modification product after the collection desalination, the concentration that records Nucleotide is 3.52ug/uL; Stand-by.
Crosslinked and the purifying of transgenosis Cry1Ac protein monoclonal antibody and Nucleotide after C, modification
To fully react 12 hours under identifying also after desalination that modifying successful antibody (i.e. antibody modification product after successful desalination is modified in judgement) evenly mixes with the 1:10 mol ratio with oligonucleotide (the nucleotide modification product after successful desalination is modified in judgement), placing 4 ℃.The group of its formation has obvious absorption peak under the 350nm wavelength, record OD
350=0.083, prove crosslinked success.
Crosslinked after product needs purifying again, it is moved into the YM-100 chimney filter the inside of Mi Libo, with the 0.1mM EDTA solution washing of pH8, centrifugal (5000 rev/mins) filter 23, to remove uncrosslinked unnecessary nucleotide monomer, back-off chimney filter centrifugal (5000 rev/mins) is collected.Product is measured light absorption value under 350nm, obtain the cross-linking products concentration after purifying, in the situation that the less and system volume of loss is consistent, front roughly the same of the crosslinked concentration after purifying and purifying, or (being 1.46mg/mL in the present embodiment) slightly on the low side.
2), antibody-Nucleotide crosslinked and microballon effect:
A, microballon are connected with complementary nucleotide
It is 0.1～0.5% that BSA is diluted to mass concentration with PBS solution (pH7.4); As washings, the coated microballon of Neutravidin is washed (totally 3 times), standing 20 minutes.With 2 * 10
-11(sequence is TCACA TTTGC CTGCA GAGAT TTCCC5 '～3 ' to the Nucleotide that mol biotin modifies, 3 ' biotin modification) add the coated magnetic bead of 5 μ l Neutravidin, 25 ℃ of shaking tables were hatched 15 minutes, with above-mentioned washings washing 3 times, obtained the coated magnetic bead of Nucleotide.
B, antibody-Nucleotide crosslinked are connected with microballon
Getting antibody-Nucleotide crosslinked 0.1 μ l(is 8.5 * 10
-13Cross-linking products after the purifying of step 1 gained of mol) add in the coated magnetic bead of above-mentioned Nucleotide, shaking table (25 ℃) was hatched 2 hours under 6 * SSPE damping fluid (pH6.0) condition, with above-mentioned washings washing 3 times.
Experiment 2, magnetic bead surfaces antibody are to the detection of transgene protein Cry1Ac
1) transgenic paddy rice being carried out transgene protein extracts.
The transgenosis test sample is obtained sample detection liquid after protein extraction and purifying.Protein extraction and purifying are known technology, can be with reference to " the extracting method and the efficient of Bt toxalbumin from transgenic paddy rice " that be published in " journal of Zhejiang university (agricultural and life science version) " 02 phase of calendar year 2001.
2) magnetic bead catching transgene protein
Transgene protein solution is added microballon system (being the magnetic bead that obtains of above-mentioned steps-antibody connector), and shaking table was hatched 1 hour under 25 ℃ of conditions.Then use PBS(pH7.4) washing, purpose is in order to wash away unconjugated transgene protein.
3) to the detection of transgene protein
With biotin labeled Cry1Ac rabbit polyclonal antibody (rabbit polyclonal antibody is by the preparation of Hangzhou Epitomics Inc., and biotin labeling is prepared by Shanghai biotech firm of friend section), add the magnetic bead system, shaking table was hatched 1 hour under 25 ℃ of conditions.Then use PBS(pH7.4) washing, purpose is in order to wash away unconjugated biotin labeling Cry1Ac rabbit polyclonal antibody.The Streptavidin of Cy3 mark is added the magnetic bead system, and shaking table is hatched 30min under 25 ℃ of conditions, uses PBS(pH7.4) washing, purpose is in order to wash away the Streptavidin of unconjugated Cy3 mark.
Observe with fluorescent microscope (Nikon, Inc) under the 510-560 excitation wavelength, be evenly distributed with the bright spot of star spot shape in the visual field.This result proof magnetic bead surfaces can successfully detect transgene protein.
At last, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. antibody and microballon method of attachment is characterized in that comprising the following steps:
1), antibody and Nucleotide are crosslinked:
The modification of A, antibody and desalination:
25～100 μ g immunoglobulin (Ig)s be diluted to concentration be after 2～8mg/mL with after SANH evenly mixes, hatch in the shaking table of 20～30 ℃ and modified 2～6 hours, the mol ratio of SANH and immunoglobulin (Ig) is 10～70:1;
With the antibody modification product of gained through washing, centrifuging, to remove unnecessary SANH; The antibody modification product after desalination;
The modification of B, Nucleotide and desalination:
Phosphoric acid buffer with pH 7.0～7.6 is diluted to 6～12 mg/mL with Nucleotide, adds SFB evenly to mix, and hatches to modify 2～6 hours in the shaking table of 20～30 ℃, and the mol ratio of SFB and Nucleotide is 10～70:1;
With the nucleotide modification product of gained through washing, centrifugal, filter, to remove unnecessary SFB; The nucleotide modification product after desalination;
Crosslinked and the purifying of antibody and Nucleotide after C, modification
Antibody modification product after desalination and the nucleotide modification product after desalination are evenly mixed according to the mol ratio of 1:8 ~ 12, fully reacted 8～24 hours in 2～8 ℃,
With the crosslinked after product of gained through washing, centrifugal, filter, remove free oligonucleotide and carry out desalination; The antibody-Nucleotide crosslinked after purifying;
2), antibody-Nucleotide crosslinked and microballon effect:
Microballon is first through the washings washing, and is then standing;
With 1～9 * 10
-11The Nucleotide that mol biotin modifies adds the coated magnetic bead of Neutravidin of 5～15 μ l, hatches 10～30 minutes in the shaking table of 20～30 ℃, then washs with washings; Add subsequently above-mentioned 5 ~ 10 * 10
-13Antibody after the mol purifying-Nucleotide crosslinked is hatched 1～4 hour in the shaking table of 20～30 ℃, then is washed with washings under 6 * SSPE damping fluid (pH 6.0) condition; Get the microballon system;
Described washings is that 0.1 ~ 0.5 % gets for BSA is diluted to mass concentration with PBS solution (pH 7.4).
2. antibody according to claim 1 and microballon method of attachment is characterized in that:
The microballon system of gained is carried out following steps:
Add the microballon system at the fluorescence two of Cy3 mark in anti-or transgene protein, shaking table was hatched 1～4 hour under 20～30 ℃ of conditions; Then with PBS(pH 7.4) wash away unconjugated fluorescence two and resist or transgene protein, use at last the fluorescence microscope result.
3. antibody according to claim 1 and 2 and microballon method of attachment is characterized in that:
In described step 1) A: the rotating speed of shaking table is 50～150 rev/mins; EDTA solution with 0.1 mM of pH 8 washs;
In described step 1) B: the rotating speed of shaking table is 50～150 rev/mins; EDTA solution washing with 0.1 mM of pH 8;
In described step 1) C: the EDTA solution washing of the crosslinked after product of gained being used 0.1 mM of pH 8.
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| CN106198963A (en) * | 2016-08-31 | 2016-12-07 | 上海美吉生物医药科技有限公司 | A kind of for immunomagnetic beads capturing leukocyte and preparation method thereof |
| CN112630422A (en) * | 2020-11-30 | 2021-04-09 | 浙江正熙生物医药有限公司 | Method for improving signal-to-noise ratio of antibody and fluorescent protein directional coupling label |
| CN114544939A (en) * | 2022-01-26 | 2022-05-27 | 天津鸿宇泰生物科技有限公司 | Streptavidin magnetic bead marking method |
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